The present invention relates to a method for identifying compounds capable of inhibiting activation by cytokines, in particular by interferon xcex3, of expression of the CIITA gene which itself is involved in controlling and regulating the expression of genes coding for MHC class II molecules.
Molecules of the major histocompatibility complex (hereinafter designated MHC) class II are transmembrane heterodimeric glycoproteins which are directly involved in the activation of CD4+ T helper lymphocytes during the immune response.
In man, this class II complex is represented by molecules belonging to the HLA (human leukocyte antigen) system. Genes coding for the xcex1 and xcex2 chains constituting the HLA-DR, HLA-DQ and HLA-DP molecules are located in region D of chromosome 6.
Expression of these genes is perfectly regulated. In contrast to genes coding for MHC class I molecules which are expressed ubiquitously, the genes coding for MHC class II molecules are expressed either constitutively uniquely in some cell types such as B lymphocytes, activated T lymphocytes, macrophages, thymic epithelium cells, or dentritic cells such as Langerhans cells, or inductively after stimulation, for example by cytokines, and more particularly by interferon xcex3 (INF xcex3) or interleukin 4 (IL4), in several other cell types such as cells from the macrophage or monocyte line, endothelial cells, fibroblasts, muscle cells or cancer cells such as melanoma cells.
Further, in B lymphocytes, the expression of genes coding for MHC class II molecules is transitory. Differentiation of B cells into plasmocytes producing immunoglobulins is accompanied by extinction of certain genes, including those coding for MHC class II.
Similarly, it has been shown that the amount of expression of MHC class II molecules constitutes a determining factor in the T cell activation process.
As a result, it is clear that molecular mechanisms for regulating the expression of these genes constitute a key element in the effectiveness of the immune response. Any defect in this regulation process can result in substantial immunological problems, or autoimmune diseases. Thus in some cases, abnormal expression of MHC class II genes has been observed on the surface of cells which normally should not express such genes. Similarly, an over-expression of these genes can be observed, leading to aberrant and uncontrolled activation of CD4+ lymphocytes (BOTTAZZO et al., 1986, Immunol. Rev., 94, 137-169). Such manifestations could be at least partially responsible for diseases such as insulin dependent diabetes, multiple sclerosis, rheumatoid arthritis or lupus erythematosus. In contrast, in some patients immunodeficiency has been demonstrated resulting from problems with expression of MHC class II genes. An example which can be cited is BLS syndrome (bare lymphocyte syndrome), which is a recessive autosomal disease in which expression of MHC class II genes is very limited or even non-existent, resulting in an absence of cellular and humoral immune response and accompanied by numerous infections which are often fatal.
A number of scientific teams have analysed the mechanisms of regulation of MHC class II gene expression and have identified a certain number of transactivating molecules which can directly or indirectly bind to specific promoter sequences of said genes (for a review, see MACH et al., 1996, Annu. Rev. Immunol. 14, 301-331).
The Applicant has previously identified and characterized one of these factors, the CIITA factor (class II transactivator) [STEIMLE et al., 1993, Cell 75, 135-146 and EP-A-0 648 836]. Further, International patent application WO-A-9606107 has shown that there are two domains in the CIITA factor which are more involved in activation of transcription of MHC class II genes. However, surprisingly and in contrast to that which has been observed for the other factors involved in regulating the expression of MHC class II genes [COGSWELL et al., 1991, Crit. Rev. Immunol. 11, 87-112], STEIMLE et al have shown that expression of the CIITA factor coincides closely with expression of MHC class II genes and is absolutely required both for constitutive expression and for induction of said MHC genes. Further, SILACCI et al (1994, J. Exp. Med., 180, 1329-1336) have shown that extinction of MHC class II genes during differentiation of plasmocytes is associated with extinction of the gene coding for the CIITA factor.
Further, LENNON et al (1997, Immunogenetics, 45, 266-273) have identified the promoter sequence of a CIITA gene which is responsible for differential expression of this factor in B cells. However, the existence of this single sequence does not explain why differential expression of the CIITA factor is observed in different cell types. Further, it does not account for induction by cytokines.
In previous studies, the Applicant used samples from different tissues of human origin to identify the complex organisation of sequences providing control of expression of the CIITA factor, the Applicant isolated and characterized several promoter regions and the Applicant demonstrated the existence of different forms of the CIITA factor and also different CIITA genes. These studies have formed the basis of a publication (MUHLETHALER-MOTTET et al., 1997, EMBO J., 16, 2851-2860) and form the subject matter of French patent application 97/04954 the contents of which are hereby incorporated into the present application. The inventors have thus shown that the different promoters identified can be activated selectively: two of the promoters are responsible for constitutive expression of the CIITA gene in dendritic cells (promoter I) and in B lymphocytes (promoter III) while promoter IV is involved in expressing the CIITA gene after induction by a cytokine, in particular interferon xcex3.
More particularly, the inventors have identified a sequence capable of expressing a transcriptional promoter activity after induction by a cytokine, such as interferon xcex3 or interleukin 4. Such a sequence is represented by the sequence comprising all or part of a sequence identified as SEQ ID NO:1 (set forth on Table 1), or its complementary sequence. An analysis of this sequence has identified several regions corresponding to cis acting regulation expression sites, such as the NF-GMa site, the GAS element, the E-box or the IRF-1 factor binding site (MUHLETHALER-MOTTET et al, 1997, EMBO J., 16, 2851-2860 and FIG. 1).
More recently, a number of studies have provided a deeper understanding of the succession of events and signals involved in activating genes expressed in response to induction by a cytokine, in particular by interferon y. An activation scheme has been proposed by DARNELL, (1997, Science 277, 1630-1635). In that model, firstly the activating cytokine, for example interferon xcex3, binds to its surface cell receptors thus enabling activation of cellular tyrosine kinases JAK1 and JAK2. Then the tyrosine residues of the STAT1 transcription factor, located in the cell cytoplasm, are phosphorylated by activated JAK kinases. This phosphorylation then enables the activated STAT1 factor to migrate into the nucleus where it binds to the GAS box of promoters inducible by cytokines (for example interferon xcex3) thus enabling activated expression of genes under the control of such promoters.
The implication of such a JAK/STAT1 activation system in the control of expression of CIITA genes inducible by interferon xcex3 has been the subject of studies which have established that, as with other genes which are inducible by interferon xcex3, expression of the CIITA factor cannot be induced in cell lines which are deficient for JAK1 (CHANG et al., 1994, J. Exp. Med., 180, 1367-1374).
Similarly, MERAZ et al. (1996, Cell, 84, 431-442) have shown that CIITA gene expression is not induced by interferon Y in bone marrow macrophages from the STAT1xe2x88x92/xe2x88x92 mouse, suggesting a determining role for STAT1 in inducing the expression of the CIITA gene by interferon xcex3.
Further, LEE and BENVENISTE (1996, J. Immunol. 157, 1559-1568) have carried out experiments using antisense oligonucleotides specific for the nucleic acid sequence coding for the STAT1 protein factor to demonstrate that the reduction in the expression of the STAT1 protein is accompanied by a reduction in the expression of the CIITA gene which can be observed after induction by interferon xcex3.
Finally, it has been shown that the STAT1 factor specifically recognises a particular nucleic acid sequence known as the xe2x80x9cGAS elementxe2x80x9d (DARNELL, 1997, Science 277, 1630-1635). An analysis of the promoter IV sequence of the CIITA gene (inducible by cytokines: MUHLETHALER-MOTTET et al., 1997, EMBO J. 16, 2851-2860xe2x80x94and FIG. 1) has revealed the presence of such a sequence.
As has been described above, promoter IV also comprises the CACGTG sequence (E-box, GREGOR et al., 1990, Genes Dev., 4, 1730-1740, see FIG. 1). This could indicate that a transcription factor belonging to the helix/loop/helix/leucine zipper family may intervene in regulating the expression of genes placed under the control of promoter IV. A number of factors from this family have been described in the literature, in particular constitutively expressed transcription factors such as TFE3 factors (BECKMANN et al., 1990, Genes Dev., 4, 167-179), USF1 (GREGOR et al., 1990, Genes Dev., 4, 1730-1740) and USF 2 (SIRITO et al., 1994, Nucleic Acid Res., 22, 427-433) or proteins involved in the Myc system such as Myc-Max or Mad-Max (AYER et al., 1993, Cell 72, 211-222).
More particularly, the USF1 transcription factor is expressed ubiquitously and participates in regulating the expression of different genes, certain of which are expressed in a xe2x80x9ctissue specificxe2x80x9d manner or in an inducible manner, for example the gene coding for the human growth hormone (PERITZ et al., 1988, J. Biol. Chem., 263, 5005-5007), the gene coding for the xcex2 chain of immunoglobulins (CHANG et al., 1992, Nucleic Acid Res., 20, 287-293) or the gene coding for p53 (REISMAN and ROTTER, 1993, Nucleic Acid Res., 21, 345-350).
The Applicant has now demonstrated that STAT1 and USF1 transcription factors respectively bind to the GAS element and to the E-box of promoter IV. However, highly surprisingly, the Applicant has also demonstrated that binding of the STAT1 factor to the GAS site is greatly stabilized by the USF1 factor and that these factors are co-operatively bound to the binding sites located on promoter IV, this co-operative interaction playing a deciding role in controlling the specific activation of promoter IV by cytokines, in particular by interferon xcex3.
The expression xe2x80x9cco-operatively boundxe2x80x9d means that there is an interaction between the protein factors in question, which may take place before or after binding to their respective site, which can define specific interaction sites between said protein factors and which result in a co-operative effect. This co-operative effect is a result which can only be observed when the interaction in question takes place; for example this co-operative effect will consist in stabilizing binding of at least one of the protein factors to its site (it being understood that this is because of the interaction existing between the protein factors). In one particular case, the co-operative effect is a synergistic effect characterized in that the effect observed using the protein factors is more than the expected effect corresponding to the sum of the individual effects observed for each of the factors.
This mechanism for inducing expression of a gene placed under the control of promoter IV is distinguished from other systems previously described for genes inducible by interferon y in that this requires the USF1 factor as an essential partner for binding and as a result for the activity of the STAT1 factor. The discovery of this mechanism, in particular involved in activating the expression of the CIITA gene by interferon xcex3, and as a result in inducing MHC class II molecules by interferon xcex3, has led the Applicant to develop a novel method for identifying molecules which are capable of inhibiting the expression of genes placed under the control of a promoter the activity of which is induced by co-operative binding of STAT1 and USF1, and more particularly under the control of all or a portion of promoter IV. Preferably, said gene is the gene coding for CIITA.
The term xe2x80x9cnucleic acid sequence coding for the CIITA polypeptidexe2x80x9d means the sequence in question comprises all or a portion of a nucleic acid sequence corresponding to mRNA from different tissues or cell lines expressing a CIITA activity in a constitutive manner or after induction. Thus they can be at least partially coding sequences or, for example, sequences involved in controlling expression, in particular sequences with a transcriptional promoter activity.
The term xe2x80x9cnucleic acid sequencexe2x80x9d means a fragment of DNA and/or RNA, double or single stranded, isolated naturally occurring, or synthetic, forming a precise concatenation of modified or non modified nucleotides, which defines a fragment or a region of a nucleic acid.
The term xe2x80x9cpolypeptidexe2x80x9d means a precise natural, isolated or synthesized, modified or non modified concatenation of amino acids, independent of its size or function.
The term xe2x80x9cnucleic acid sequence with a transcriptional promoter activityxe2x80x9d means a nucleic acid sequence which can control i.e., initiate and/or modulate, transcription of at least one homologous or heterologous gene located downstream of said sequence. Similarly, the promoter function of said sequences or promoter will be mentioned.
The term xe2x80x9creporter genexe2x80x9d means any nucleic acid sequence located downstream of a second nucleic acid sequence, permitting the analysis of the transcriptional promoter activity of said second sequence. In effect, transcription of that reporter gene results in the appearance of a product (RNA or polypeptide) which is readily detectable using well known conventional techniques.
The term xe2x80x9cSTAT1 transcription factor or polypeptidexe2x80x9d means the STAT1 transcription factor which is capable of binding to the GAS element of promoters which are inducible by interferon xcex3 (DARNELL, 1997, Science 277, 1630-1635).
The term xe2x80x9cUSF1 transcription factor or polypeptidexe2x80x9d means the USF1 transcription factor which is capable of binding to the E-box of promoters such as the promoter of the gene coding for human growth hormone (PERITZ et al., 1988, J. Biol. Chem. 263, 5005-5007), the gene coding for the immunoglobulin xcex2 chain (CHANG et al., 1992, Nucleic Acid Res., 20, 287-293) or the gene coding for p53 (REISMAN and ROTTER, 1993, Nucleic Acid Res., 21, 345-350).
It should also be mentioned that said STAT1 and USF1 factors can be recombinant or natural in origin, and more particularly consist of factors available in cell extracts, in particular nuclear extracts, prepared from cell lines, possibly stimulated by a cytokine, in particular interferon xcex3, expressing said factors. More particularly regarding the STAT1 factor, this can be either in a non activated form (non phosphorylated) or in an activated form (phosphorylated, in particular by the action of the JAK1 enzyme). Within the context of the present invention, the activated form of STAT1 is preferably selected.